The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

Norwalk virus capsid protein was produced by expression of the second and third open reading frames of the Norwalk virus genome, using a cell-free translation system and baculovirus recombinants. Analysis of the expressed products showed that the second open reading frame encodes a protein with an apparent molecular weight of 58,000 (58K protein) and that this protein self-assembles to form empty viruslike particles similar to native capsids in size and appearance. The antigenicity of these particles was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assays of paired serum samples from volunteers who developed illness following Norwalk virus challenge. These particles also induced high levels of Norwalk virus-specific serum antibody in laboratory animals following parenteral inoculation. A minor 34K protein was also found in infected insect cells. Amino acid sequence analysis of the N terminus of the 34K protein indicated that the 34K protein was a cleavage product of the 58K protein. The availability of large amounts of recombinant Norwalk virus particles will allow the development of rapid, sensitive, and reliable tests for the diagnosis of Norwalk virus infection as well as the implementation of structural studies.

Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.

Pandemic influenza virus has its origins in avian influenza viruses. The highly pathogenic avian influenza virus subtype H5N1 is already panzootic in poultry, with attendant economic consequences. It continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. Therefore, H5N1 virus has rightly received attention as a potential pandemic threat. However, it is noted that the pandemics of 1957 and 1968 did not arise from highly pathogenic influenza viruses, and the next pandemic may well arise from a low-pathogenicity virus. The rationale for particular concern about an H5N1 pandemic is not its inevitability but its potential severity. An H5N1 pandemic is an event of low probability but one of high human health impact and poses a predicament for public health. Here, we review the ecology and evolution of highly pathogenic avian influenza H5N1 viruses, assess the pandemic risk, and address aspects of human H5N1 disease in relation to its epidemiology, clinical presentation, pathogenesis, diagnosis, and management.

http://www.hkupasteur.hku.hk/publications/pdf/Peiris_et_al_2007.pdf

Avian Influenza Virus (H5N1): a Threat to Human Health

The biology of influenza viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.

https://cmr.asm.org/content/cmr/14/1/129.full.pdf

Pandemic Threat Posed by Avian Influenza A Viruses

Ten serologically distinct avian influenza A viruses were administered to squirrel monkeys and hamsters to compare their replication and virulence with those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the 10 avian influenza A viruses exhibited a spectrum of replication and virulence. The levels of virus replication and clinical response were closely correlated. Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8), resembled the human virus in their level and duration of replication and in their virulence. At the other end of the spectrum, five avian viruses were restricted by 100- to 10,000-fold in replication in the upper and lower respiratory tract and were clearly attenuated compared with the human influenza virus. In hamsters, the 10 viruses exhibited a spectrum of replication in the nasal turbinates, ranging from viruses that replicated as efficiently as the human virus to those that were 8,000- fold restricted. Since several avian viruses were closely related serologically to human influenza viruses, studies were done to confirm the avian nature of these isolates. Each of the avian viruses plaqued efficiently at 42°C, a restrictive temperature for replication of human influenza A viruses. Avian strains that had replicated either very efficiently or very poorly in squirrel monkeys still grew to high titer in the intestinal tracts of ducks, a tropism characteristic of avian, but not mammalian, influenza viruses. These observations indicate that some avian influenza A viruses grow well and cause disease in a primate host, whereas other avian viruses are very restricted in this host. These findings also provide a basis for determining the gene or genes involved in the restriction of replication that is observed with the attenuated avian viruses. Application of such information may allow the preparation of reassortant viruses derived from a virulent human influenza virus and an attenuated avian virus for possible use in a live attenuated vaccine for prevention of influenza in humans.

Virulence of Avian Influenza A Viruses for Squirrel Monkeys

A fecal filtrate of human origin containing the Norwalk agent of epidemic viral gastroenteritis was administered by stomach tube to chimpanzees in an attempt to induce diarrheal disease. Significant postchallenge serum antibody rises against Norwalk viral antigens were demonstrated in all animals using the techniques of immune electron microscopy and radioimmunoassay. In addition, viral antigens were detected in feces from five of nine animals using radioimmunoassay. Clinical illness characterized by diarrhea and/or vomiting did not occur. Infection was transmitted subsequently by feeding four additional chimpanzees a fecal filtrate prepared from one of the previously infected animals. Development of an antibody response in four animals and detection of viral antigen in two animals that received this passage filtrate indicated that viral replication had occurred in the absence of clinical illness. The availability of the chimpanzee as an experimental animal host susceptible to infection with the Norwalk agent should facilitate the study of epidemic viral gastroenteritis.

Experimental infection of chimpanzees with the Norwalk agent of epidemic viral gastroenteritis

Into 14 juvenile cebus monkeys that lacked serum antibodies for RS virus 10(8) plaque-forming units (pfu) of wild-type respiratory syncytial (RS) virus were inoculated transtracheally. Roentgenographic evidence of pneumonia developed in 13 of 14 infected animals. Gross pathologic changes occurred in each of the 13 monkeys that were sacrificed. Patchy areas of red consolidation were seen in the lower lobes 24 hours after inoculation, and there was progression to gray consolidation seven days later. Each of the infected animals had histologic evidence of interstitial pneumonia. Changes were detected in the lung as early as 24 hours after inoculation; they consisted primarily of infiltration of the alveolar wall. By the fourth to sixth day after inoculation there was marked interstitial thickening, pulmonary consolidation, formation of multinucleated giant cells and development of eosinophilic cytoplasmic inclusion bodies within alveolar cells. RS viral antigens, detected by indirect immunofluorescence, were distributed throughout cells of the alveolar wall and the bronchiolar epithelium. The virus grew to highest titer in the lungs on the fourth to sixth day after inoculation; up to 10(8) pfu/gram of tissue were detected. The cebus monkey represents the first experimental host to develop extensive pulmonary lesions during infection with respiratory syncytial virus.

Experimental respiratory syncytial virus pneumonia in cebus monkeys.

Approximately 104 plaque-forming units (pfu) of five temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus were administered intranasally to seronegative chimpanzees to evaluate their level of attenuation. When the ts-1 mutant was given to two chimpanzees, the pattern of viral growth and illness (rhinorrhea) was similar to that seen during wild-type RS virus infection. Two further defective subclones of ts-1, ts-1 NG-1 and ts-1 NG-16, appeared more attenuated than ts-1 with respect to severity of rhinorrhea. The ts-7 mutant infected the one animal tested; illness was similar to that induced by ts-1 or wild-type virus. 104 pfu of the ts-2 mutant failed to infect two chimpanzees; virus was not isolated, a serum antibody response was not detected, and unlike chimpanzees given the other ts mutants, these animals were not resistant to challenge with wild-type virus. A 40-fold larger inoculum of ts-2, 105, 6pfu, infected each of four chimpanzees, but the peak quantity of virus shed was approximately 500-fold lower than during wild-type virus infection. Significantly, the ts-2 infected chimpanzees did not become ill, but infection did stimulate the development of RS virus serum neutralizing antibodies. The one chimpanzee given 107, 6 pfu of the ts-2 mutant shed as much virus as the chimpanzees infected with wild-type virus; however, there were no signs of upper respiratory tract illness. Owl monkeys resembled chimpanzees in shedding s large amount of virus and developing rhinorrhea following administration of wild-type virus or the ts-1 mutant. Significantly, 105, 3 pfu of ts-2 infected only one of two owl monkeys tested, but a small quantity of virus (>10 pfu/ml) was shed. These observations suggested that the ts-2 mutant was the most attenuated vaccine cadidate strain tested, and that extent of infection with rs-2 appeared to be related to the quantity of virus administered.

Evaluation of five temperature-sensitive mutants of respiratory syncytial virus in primates: I. Viral shedding, immunologic response, and associated illness.

Five temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus (ts-1, ts-1 NG-1, ts-1 NG-16, ts-2, and ts-7), previously evaluated forinfectivity and virulence in chimpanzees and owl monkeys, were also assayed for in vivo genetic stability. None of the five mutants tested was completely stable genetically. Thus, virus which had lost some or all of the ts property was recovered from each infected chimpanzee. Significantly, each ts-1 NG-1 isolate retained some degree of temperature sensitivity and hence was not true wild-type virus. Clonal analysis of viruses shed by ts-1, ts-1 NG-1, ts-1 NG-16, or ts-7 infected chimpanzees indicated that in most instances only a minority of the virus shed was altered genetically. Of five chimpanzees infected with the ts-2 mutant, three shed only ts virus, and the remaining two chimpanzees shed only ts+ virus. Such ts+ virus proved to be avirulent when evaluated in chimpanzees or owl monkeys, indicating that loss of the ts property did not restore virulence. Based upon these findings, the ts-2 mutant appears to be a suitable candidate for clinical trials in man.

D. Lewis Sly

Papers

Virulence of Avian Influenza A Viruses for Squirrel Monkeys

Experimental infection of chimpanzees with the Norwalk agent of epidemic viral gastroenteritis

Experimental respiratory syncytial virus pneumonia in cebus monkeys.

Evaluation of five temperature-sensitive mutants of respiratory syncytial virus in primates: I. Viral shedding, immunologic response, and associated illness.

Evaluation of five temperature-sensitive mutants of respiratory syncytial virus in primates: II. Genetic analysis of virus recovered during infection.